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Laemmllie buffer composition for diluting protein
Posted by: Raju (IP Hidden, Unregistered user, )
Date: February 17, 2005 11:25AM
Hi there,
Does anybody knows the composition of laemmlli buffer for diluting and storing Myofibrillar proteins? If anybody knows please let me know the composition and what concentration would be recommendable for diltuing the sample, what is the best dilution ratio. I do have one more doubt. Can we measure preotein conc by bradford assay after diltung protein sample in laemmlli buffer? Please give me some ideas Thanks Raju
Re: Laemmllie buffer composition for diluting protein
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: March 9, 2005 01:48PM
I know the standard Laemmlli formula, but am uncertain of a specific one for the proteins you mentioned. The standard formula is: 62.5 mM Tris-HCL, pH 6.8; 2% SDS, 25% glycerol, 0.01% bromophenol blue. The dilution ratio normally is 1:2. This buffer would not be appropriate for protein dilution prior to performing the bradford assay, which requires an SDS concentration of less than 0.1% to prevent interferrence. Perhaps you could try diluting the proteins in something else* or simply leave the SDS out of the buffer until after measurement. You could then add the SDS shortly before running western blots to denature the proteins. Most Lammelli buffers will contain a higher concentration of SDS than the bradford assay allows. *We dilute our proteins in 0.1% SDSand store them at -70C. They can be kept this way for at least one year. You may want to try another protein assay, perhaps the BCA method which can tolerate an SDS concentration of 5% without interferrence. Hope this helps. :)
Re: Laemmllie buffer composition for diluting protein
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: March 14, 2005 12:37PM
The Laemmili buffer formula used for storing you rprotein should not have bromophenol blue as it will make protein determination with BCA or any detergent tolerant protein concentratin method quite difficult. However, you can always add this stain to your sample after measuring its protein concentration where using BCA is an excellent suggestion.
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